Cytological system illumination integrity checking apparatus and method

ABSTRACT

A method for checking cytological system illumination including the steps of checking global illumination variation, static field uniformity, dynamic field uniformity, specimen thickness variation, strobe repeatability, calibration plate cleanliness, and strobe dropout. A calibration plate and test target is employed for various illumination checks.

This application is a division of application Ser. No. 08/303,179, filedSep. 8, 1994, now U.S. Pat. No. 5,715,326.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and apparatus for evaluationof temporal and spatial variation of illumination in automated machinevision instruments. More specifically, the evaluation is conducted tocharacterize variations in global illuminance, static field intensity,dynamic field intensity, strobe repeatability, and intensity due tofactors such as, for example, specimen thickness and cleanliness ofsystem calibration hardware. Still more particularly, the presentinvention relates to automated instruments used for analyzing biologicalor cytological specimens, such as sputum samples, urine samples or papsmears.

2. Discussion of the Prior Art

Automated analysis of biological specimens requires a high degree ofspatial and temporal uniformity for accurate and repeatable evaluation.Those evaluations often measure photometric properties such as nuclearand cytoplasm optical density. In order to accurately and repeatablymeasure these properties, the illumination must maintain a high degreeof uniformity across the field of view and from collected image tocollected image. In addition, morphological operations are conducted tosegment various objects in the field of view for further analysis todetermine various feature values related to size, shape and frequencycontent, among other factors. Operations used in such analyses tend toexhibit nonlinear behavior due to various thresholding schemes that maybe employed. Therefore, accurate and repeatable behavior of theseprocessing methods also necessitates a high degree of uniformity acrosseach field of view and from collected image to collected image.

SUMMARY OF THE INVENTION

The present invention provides a method for checking system illuminationin an automated cytological system comprising the steps of checkingglobal illumination variation, static field uniformity, dynamic fielduniformity, specimen thickness variation, strobe repeatability,calibration plate cleanliness, and strobe dropout.

It is an object of this invention to provide a means to characterizeglobal illumination variation.

It is an object of this invention to provide a means to characterizestatic field uniformity.

It is an object of this invention to provide a means to characterizedynamic field uniformity.

It is an object of this invention to provide a means to characterizefield uniformity variations due to specimen thickness. As used hereinspecimen thickness refers to the combined thickness of the substrate,objects of interest, mounting media and coverslip.

It is an object of this invention to provide a means to characterizestrobe repeatability.

It is an object of this invention to provide a means to characterizecleanliness of system calibration hardware.

It is an object of this invention to provide a means to characterize anacceptable amount of strobe dropout behavior.

It is an object of this invention to provide a means to characterize andevaluate the acceptability of illumination for automated cytologicalanalysis.

It is an object of this invention to provide a runtime means tocharacterize and evaluate the acceptability of illumination forautomated cytological analysis.

It is an object of this invention to provide a runtime means tocharacterize and evaluate the acceptability of illumination for anautomated vision interpretation system for the analysis of pap smears.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art through the description ofthe preferred embodiment, claims and drawings herein wherein likenumerals refer to like elements.

BRIEF DESCRIPTION OF THE DRAWINGS

To illustrate this invention, a preferred embodiment will be describedherein with reference to the accompanying drawings.

FIGS. 1A, 1B, 1C and 1D show an automated cytology system and theplacement of a calibration and test target into an optical path of anautomated microscope as employed by the method and apparatus of theinvention.

FIG. 2 shows a histogram of global illumination variation.

FIG. 3 shows an illustration of two illuminated fields as may beevaluated by a dynamic field uniformity test employed by the method ofthe invention.

FIG. 4 shows an evaluation apparatus used in a strobe repeatability testmethod of the invention.

FIG. 5 shows a high level flow diagram of one example of the method ofthe invention to check cytological system illumination integrity.

FIG. 6 shows a flow diagram of one example of a global illumination testas provided by the method of the invention.

FIG. 7 shows a flow diagram of one example of a method for checkingstatic field of uniformity as employed by the method of the invention.

FIG. 8 shows a flow diagram of one method of the invention for checkingillumination variation due to specimen thickness variation.

FIG. 9 shows a flow diagram of one method of the invention for checkingstrobe repeatability.

FIG. 10 shows a flow diagram of one method of the invention for checkingcalibration plate cleanliness.

FIG. 11 shows a flow diagram of one method of the invention for checkingstrobe drop out.

FIG. 12 shows a flow diagram of one method of the invention for checkingdynamic field uniformity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Now refer to FIGS. 1A and 1B which show a schematic diagram of oneembodiment of the apparatus of the invention for checking illuminationintegrity for an automated microscope. While the method and apparatus ofthe invention will be discussed in terms of an example herein related toan automated cytology apparatus, it will be understood that theinvention is not so limited. The features and principles of theinvention may be applied to check urine analysis processes,semiconductor process defects, liquid crystal devices and other types ofprocessing systems employing, for example, continuous arc lamps,filament lamps, laser sources, tube cameras, PIN diodes andphotomultiplier tubes.

The apparatus of the invention comprises an imaging system 502, a motioncontrol system 504, an image processing system 536, a central processingsystem 540, and a workstation 542. The imaging system 502 is comprisedof an illuminator 508, imaging optics 510, a CCD camera 512, anillumination sensor 514 and an image capture and focus system 516. Theimage capture and focus system 516 provides video timing data to the CCDcameras 512, the CCD cameras 512 provide images comprising scan lines tothe image capture and focus system 516. An illumination sensor intensityis provided to the image capture and focus system 516 where anillumination sensor 514 receives the sample of the image from the optics510. In one embodiment of the invention, the optics may further comprisean automated microscope. The illuminator 508 provides illumination of aslide. The image capture and focus system 516 provides data to a VME bus538. The VME bus distributes the data to an image processing system 536.The image processing system 536 is comprised of field-of-view processors568. The images are sent along the image bus 564 from the image captureand focus system 516. A central processor 540 controls the operation ofthe invention through the VME bus 538. The central processor includes ahost CPU 562. In one embodiment the host CPU 562 comprises a Motorola68030 CPU. The motion controller 504 is comprised of a tray handler 518,a microscope stage controller 520, a microscope turret 522, and acalibration slide 524. The motor drivers 526 position the slide underthe optics. A bar code reader 528 reads a barcode located on thecalibration slide 524. A touch sensor 530 determines whether a slide isunder the microscope objectives, and a door interlock 532 preventsoperation in case the doors are open. Motion controller 534 controls themotor drivers 526 in response to the central processor 540. An Ethernet(™) communication system 560 communicates to a workstation 542 toprovide control of the system. A hard disk 544 is controlled byworkstation 550. In one embodiment, workstation 550 may comprise a SunSparc Classic (™) workstation. A tape drive 546 is connected to theprocessor 550 as well as a modem 548, a monitor 552, a keyboard 554, anda mouse pointing device 556. A printer 558 is connected to the Ethernet(™) network 560.

During illumination integrity checking, the central computer 540,running a real time operating system, controls the microscope and theprocessor to acquire and digitize images from the microscope. Theflatness of the slide may be checked, for example, by contacting thefour corners of the slide using a computer controlled touch sensor. Thecomputer 540 also controls the microscope stage to position the specimenunder the microscope objective, and from one to 15 field of view (FOV)processors 568 which receive images under control of the computer 540.

Referring now to FIGS. 1C and 1D, there shown is placement of acalibration and test target 1 into an optical path of an automatedmicroscope 3. Several of the processes employed by the present inventionrequire such a calibration and target plate. In the case of atransmission microscope, the calibration and test target 1 may be aclear piece of glass that is approximately 1.45 mm thick. Thecalibration and test target advantageously comprises specified clearareas and image primitives such as horizontal and vertical bar targets.Such calibration and test target plates are used for most transmissionmicroscopes to simulate the optical path difference effects introducedby the substrate, coverslip and specimen media. The calibration and testtarget 1 is positioned longitudinally away from a plane of best focus 2to reduce the effects of flaws in the glass and contaminants 4 that maystick to the surface of the calibration and test target plate.

Referring now to FIG. 5, a flow diagram of one example of the method ofthe invention for cytological system illumination integrity checking isshown. The order of checking process steps shown in FIG. 5 is by way ofillustration only and is not intended to so limit the invention. Thecytological system illumination integrity checking method 100 includeschecking global illumination variation at process step 10, checkingstatic field uniformity at process step 12, checking dynamic fielduniformity at process step 14, checking specimen thickness variation atprocess step 16, checking strobe repeatability at process step 18,checking calibration plate cleanliness at process step 20 and checkingstrobe dropout at process step 22. Each of these process steps will beexplained in more detail hereinbelow.

Global Illumination

Illumination sources typically employed in automated vision analysisinstruments exhibit variations in energy output from one collected imageto another collected image. In the case of pulsed arc lamps, the arc isunconstrained and can vary spatially between flashes. The combinedeffects of energy output variations and spatial variations may cause avariation of illuminance over the entire field of view from collectedimage to collected image. In addition, in order to approach optimal useof an imaging device, such as a CCD focal plane, the imaging deviceshould be used as near as possible to its optimal dynamic range. That isto say, illuminance should be set at a level suitable for obtaining theoptimal discrimination performance of the imaging device for theapplication.

Referring now to FIG. 6, a flow diagram of one method of the inventionfor providing global illumination variation tests 10 is shown. Globalillumination tests are used to check the illuminance level of a lightsource, such as an arc lamp. A calibration and test target is introducedinto the optical path at process step 60 and positioned at process step62. At process step 63, the system illuminance is adjusted to an optimallevel as may best be determined by an operator or automated system, forexample. A predetermined number of images are acquired at process step64. In one example of the invention, about one hundred (100) images areacquired. Each image may comprise a 512×512 array of pixels that are 256grey levels deep. The mean pixel value for each acquired image iscomputed and temporarily stored at process step 65. These mean intensityvalues are tabulated at step 66 in a histogram format like that shown inTable 1. The left most column of Table 1 represents the mean intensityof the illuminated field in counts. The right most column denotes thenumber of occurrences of the corresponding mean field intensity for theone hundred images acquired. The center column represents a normallydistributed variation with a mean and standard deviation similar to theactual data. It is only shown to demonstrate that the actual data variesin accordance with normally distributed population. Therefore, the datacan be analyzed using standard statistical parameters.

FIG. 2 shows a plot of the data in Table 1. It is apparent from FIG. 2that the variation behaves like a normal random distribution. Themaximum, minimum, mean and coefficient of variation for the distributionare determined at process step 67. A coefficient of variation of thesemean values is determined at process step 68. These parameters arecompared, in step 69 against engineered limits as shown in Table 2.Camera dynamic range is defined herein as the mean of mean voltages forall images. Examples of engineering limits used in one example of theinvention for checking global illumination variation are as follows.

    ______________________________________                                        Camera dynamic range:                                                                          0.665 V < x < 0.690 V                                        Coefficient of variation:                                                                          <1.0%                                                    Maximum % Variation:                                                                                    (max - min)/(2*mean) < 1.5%                         Mean:                                    223 counts < ∞ < 242           ______________________________________                                                         counts                                                   

                  TABLE 1                                                         ______________________________________                                        Intensity    Normal Distribution                                                                        Actual Data                                         ______________________________________                                        226          0.00         0                                                   227                       0.00                                                                                                0                             228                       0.00                                                                                                0                             229                       0.00                                                                                                0                             230                       0.00                                                                                                0                             231                       0.06                                                                                                0                             232                       0.53                                                                                                0                             233                       3.06                                                                                                0                             234                       10.67                                                                                              14                             235                       22.59                                                                                              23                             236                       29.00                                                                                              29                             237                       22.59                                                                                              19                             238                       10.67                                                                                              15                             239                       3.06                                                                                  0                                           240                       0.53                                                                                                0                             241                       0.06                                                                                                0                             242                       0.00                                                                                                0                             243                       0.00                                                                                                0                             244                       0.00                                                                                                0                             245                       0.00                                                                                                0                             246                       0.00                                                                                                0                             ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Parameter Results and Limits Comparison                                       Parameter Description                                                                      Actual Value Limits                                              ______________________________________                                        Mean Intensity                                                                             236          223 counts < ∞ < 242                                                            counts                                      Coefficient of Variation                                                                       0.53%       <1.0%                                            Maximum % variation                                                                                 0.85%                                                                                (max - min)/                                                                       (2*mean) < 1.5%                             ______________________________________                                    

The method of the invention for checking global illumination ensures thefield of view is illuminated at the proper intensity and that the globalintensity does not vary by more than the limits shown.

Static Field Uniformity

Referring now to FIG. 7, a flow diagram illustrating the method of theinvention for checking static field of uniformity as employed by themethod of the invention is shown. The field of view illuminated inautomated vision analysis instruments can vary in intensity at variouspoints in the field due to misalignments, debris on optical surfaces,poor optical design in addition to other factors. In addition, thespatial response of the detector, such as a CCD camera, may also vary,exhibiting a behavior known as patterning. The totality of thesevariations may be referred to as static field uniformity.

As before, a calibration and test target is introduced into the opticalpath at process step 72. A single image is captured and a histogram isgenerated for the image pixels at process steps 73 and 74 respectively.The maximum, minimum values and the coefficient of variation of thepixels are determined for the histogram at step 75. Additionally, atstep 76, the maximum and minimum variation is computed for 99.9% of thefield-of-view by ignoring the 0.1% outlying pixels at the tails of thehistogram. This value provides a measure of the field uniformity withoutthe affects of stray pixels. The variation factors are evaluated bycomparing the coefficient of variation against a first predeterminedlimit 77, comparing the maximum variation against a second predeterminedlimit 78, comparing the partial field maximum variation against a thirdpredetermined limit 79.

                                      TABLE 3                                     __________________________________________________________________________    Intensity Histogram of Entire Field                                           Int                                                                              # Int                                                                              # Int                                                                              # Int                                                                              # Int                                                                              # Int                                                                              # Int                                                                              # Int                                                                              #                                       __________________________________________________________________________    0: 0 32:                                                                              0 64:                                                                              0 96:                                                                              0 128:                                                                             0 160:                                                                             0 192:                                                                             0 224:                                                                             2                                       1: 0   33:                                                                             0                                                                               65:                                                                             0   97:                                                                            0  129:                                                                            0  161:                                                                            0  193:                                                                            0  225:                                                                            7                                       2: 0   34:                                                                             0                                                                               66:                                                                             0   98:                                                                            0  130:                                                                            0  162:                                                                            0  194:                                                                            0  226:                                                                            213                                     3: 0   35:                                                                             0                                                                               67:                                                                             0   99:                                                                            0  131:                                                                            0  163:                                                                            0  195:                                                                            0  227:                                                                            1424                                    4: 0   36:                                                                             0                                                                               68:                                                                             0  100:                                                                            0  132:                                                                            0  164:                                                                            0  196:                                                                            0  228:                                                                            7803                                    5: 0   37:                                                                             0                                                                               69:                                                                             0  101:                                                                            0  133:                                                                            0  165:                                                                            0  197:                                                                            0  229:                                                                            47942                                   6: 0   38:                                                                             0                                                                               70:                                                                             0  102:                                                                            0  134:                                                                            0  166:                                                                            0  198:                                                                            0  230:                                                                            60366                                   7: 0   39:                                                                             0                                                                               71:                                                                             0  103:                                                                            0  135:                                                                            0  167:                                                                            0  199:                                                                            0  231:                                                                            74350                                   8: 0   40:                                                                             0                                                                               72:                                                                             0  104:                                                                            0  136:                                                                            0  168:                                                                            0  200:                                                                            0  232:                                                                            51837                                   9: 0   41:                                                                             0                                                                               73:                                                                             0  105:                                                                            0  137:                                                                            0  169:                                                                            0  201:                                                                            0  233:                                                                            15630                                   10:                                                                              0  42:                                                                              0                                                                               74:                                                                             0  106:                                                                            0  138:                                                                            0  170:                                                                            0  202:                                                                            0  234:                                                                            2231                                    11:                                                                              0  43:                                                                              0                                                                               75:                                                                             0  107:                                                                            0  139:                                                                            0  171:                                                                            0  203:                                                                            0  235:                                                                            317                                     12:                                                                              0  44:                                                                              0                                                                               76:                                                                             0  108:                                                                            0  140:                                                                            0  172:                                                                            0  204:                                                                            0  236:                                                                            22                                      13:                                                                              0  45:                                                                              0                                                                               77:                                                                             0  109:                                                                            0  141:                                                                            0  173:                                                                            0  205:                                                                            0  237:                                                                            0                                       14:                                                                              0  46:                                                                              0                                                                               78:                                                                             0  110:                                                                            0  142:                                                                            0  174:                                                                            0  206:                                                                            0  238:                                                                            0                                       15:                                                                              0  47:                                                                              0                                                                               79:                                                                             0  111:                                                                            0  143:                                                                            0  175:                                                                            0  207:                                                                            0  239:                                                                            0                                       16:                                                                              0  48:                                                                              0                                                                               80:                                                                             0  112:                                                                            0  144:                                                                            0  176:                                                                            0  208:                                                                            0  240:                                                                            0                                       17:                                                                              0  49:                                                                              0                                                                               81:                                                                             0  113:                                                                            0  145:                                                                            0  177:                                                                            0  209:                                                                            0  241:                                                                            0                                       18:                                                                              0  50:                                                                              0                                                                               82:                                                                             0  114:                                                                            0  146:                                                                            0  178:                                                                            0  210:                                                                            0  242:                                                                            0                                       19:                                                                              0  51:                                                                              0                                                                               83:                                                                             0  115:                                                                            0  147:                                                                            0  179:                                                                            0  211:                                                                            0  243:                                                                            0                                       20:                                                                              0  52:                                                                              0                                                                               84:                                                                             0  116:                                                                            0  148:                                                                            0  180:                                                                            0  212:                                                                            0  244:                                                                            0                                       21:                                                                              0  53:                                                                              0                                                                               85:                                                                             0  117:                                                                            0  149:                                                                            0  181:                                                                            0  213:                                                                            0  245:                                                                            0                                       22:                                                                              0  54:                                                                              0                                                                               86:                                                                             0  118:                                                                            0  150:                                                                            0  182:                                                                            0  214:                                                                            0  246:                                                                            0                                       23:                                                                              0  55:                                                                              0                                                                               87:                                                                             0  119:                                                                            0  151:                                                                            0  183:                                                                            0  215:                                                                            0  247:                                                                            0                                       24:                                                                              0  56:                                                                              0                                                                               88:                                                                             0  120:                                                                            0  152:                                                                            0  184:                                                                            0  216:                                                                            0  248:                                                                            0                                       25:                                                                              0  57:                                                                              0                                                                               89:                                                                             0  121:                                                                            0  153:                                                                            0  185:                                                                            0  217:                                                                            0  249:                                                                            0                                       26:                                                                              0  58:                                                                              0                                                                               90:                                                                             0  122:                                                                            0  154:                                                                            0  186:                                                                            0  218:                                                                            0  250:                                                                            0                                       27:                                                                              0  59:                                                                              0                                                                               91:                                                                             0  123:                                                                            0  155:                                                                            0  187:                                                                            0  219:                                                                            0  251:                                                                            0                                       28:                                                                              0  60:                                                                              0                                                                               92:                                                                             0  124:                                                                            0  156:                                                                            0  188:                                                                            0  220:                                                                            0  252:                                                                            0                                       29:                                                                              0  61:                                                                              0                                                                               93:                                                                             0  125:                                                                            0  157:                                                                            0  189:                                                                            0  221:                                                                            0  253:                                                                            0                                       30:                                                                              0  62:                                                                              0                                                                               94:                                                                             0  126:                                                                            0  158:                                                                            0  190:                                                                            0  222:                                                                            0  254:                                                                            0                                       31:                                                                              0  63:                                                                              0                                                                               95:                                                                             0  127:                                                                            0  159:                                                                            0  191:                                                                            0  223:                                                                            0  255:                                                                            0                                       __________________________________________________________________________     INT = INTENSITY,                                                              # = NUMBER OF PIXELS AT CORRESPONDING INTENSITY                          

                  TABLE 4                                                         ______________________________________                                        Parameter Description                                                                              Actual Value                                                                            Limits                                         ______________________________________                                        Coefficient of variation:                                                                          0.6%      <1.0%                                          Maximum % variation (full field):                                                                      2.6%     <6.0%                                       Maximum % Variation (partial field):                                                                1.7%        <3.0%                                       ______________________________________                                    

Dynamic Field Uniformity

In addition to static non-uniformity, the illumination may varydynamically from image to image. For example, on a given strobe flash,the field may have a maximum static variation of 15 units. On asubsequent flash, the maximum static variation may be also be 15 units.However, that variation may be evident in the opposite direction,resulting in an actual non-uniformity of 30 units.

FIG. 3 shows an illustration of two illuminated fields as may beevaluated by a dynamic field uniformity test employed by the method ofthe invention. In each of the two fields 302, 303 the pixel on the left304, 305 respectively, maintained a value of 230. However, the intensityof the pixel on the right 307, 308 respectively differs by 30 countsbetween the fields. This represents a change in intensity of ±6.5%. Thedynamic field uniformity test is run to check for such a condition.

FIG. 12 shows a flow diagram of one method of the invention for checkingdynamic field uniformity. As before, a calibration and test target isintroduced into the optical path in step 1202. Fifty images arecollected in step 1203 and a histogram is generated for each image instep 1204. The maximum values, minimum values and the coefficient ofvariation are determined in step 1205 for each illuminated field.Additionally, the maximum and minimum variation is computed for 99.9% ofthe field-of-view by ignoring the 0.1% outlying pixels at the tails ofthe histogram. A difference is taken between the highest and lowestpixel values for all 50 images in step 1204. This value is divided bytwo times the mean to determine the +/- full field variations in step1208. The same method is used to determine the +/- partial fieldvariation in step 1210. These values are compared against limits asshown in Table 5 in step 1212. Likewise, in step 1214 the worst, that isthe highest, coefficient of variation of the fifty fields is comparedagainst the limit shown in Table 5. Table 5 also shows values from anactual test.

                  TABLE 5                                                         ______________________________________                                        Parameter Results and Limits                                                  Comparison for Dynamic Field Uniformity                                       Parameter Description                                                                              Actual Value                                                                            Limits                                         ______________________________________                                        Coefficient of variation                                                                           0.6%      <1.0%                                          Maximum % variation (full field):                                                                       2.8%       <8.0%                                    Maximum % variation (partial field):                                                                 1.9%          <4.0%                                    ______________________________________                                    

Specimen Thickness Variation

The illumination system of FIG. 1C, for example, is designed to operatewith prescribed optical path distances. However, in transmitted lightmicroscopy, specimens are usually mounted on a substrate and a coverslipis placed over the substrate. The thickness of the substrate or slide,coverslip and mounting media may vary. These variations introduce achange in the optical path of the illumination system. These changes, ifnot carefully designed for, may degrade the illumination uniformity. Inaddition, uniformity may degrade at the upper or lower end of the slidethickness range due to the same reasons as mentioned in the static fielduniformity section. Although this degradation may occur at one or theother or both extremes, it may not occur at the nominal slide thickness.Therefore, illumination uniformity must be checked at the extremes ofthe designed operating limits. In this embodiment the system is designedto accommodate a range of 1.0 mm to 1.9 mm of combined slide, mountingmedia and coverslip thickness variations.

FIG. 8 shows a flow diagram of one method of the invention for checkingspecimen thickness variation. A calibration and test target isintroduced into the optical path 82. However, this time different partsof the calibration plate are used. The calibration plate also containsclear areas that are 1.0 mm and 1.90 mm thick. These areas are usedcorrespondingly to conduct the thickness test. In this test the staticfield uniformity test, described above is run twice--once with a 1.0 mmthick slide and a second time with a 1.9 mm thick slide at process steps84 and 86 respectively. The coefficient of variation and maximumvariation, for both the full and partial fields, are recorded for eachslide thickness 88. A second coefficient of variation and a secondmaximum variation, for both the full and partial fields, are recordedfor the second slide thickness 182. The results are evaluated bycomparing the first coefficient of variation against a firstpredetermined limit 184, comparing the first maximum variation against asecond predetermined limit 186, comparing the first maximum partialfield variation against a third predetermined limit 188, comparing thesecond coefficient of variation against a fourth predetermined limit282, comparing the second maximum variation against a fifthpredetermined limit 284, and comparing the second maximum partial fieldvariation against a sixth predetermined limit 286.

In one embodiment of the invention the following limits were employed.

                  TABLE 6                                                         ______________________________________                                        Parameter Results and Limits                                                  Comparison for Thin Slide Field Uniformity                                    Parameter Description                                                                              Actual Value                                                                            Limits                                         ______________________________________                                        Coefficient of variation:                                                                          0.6%      <1.0%                                          Maximum % variation (full field):                                                                     2.6%       <6.0%                                      Maximum % Variation (partial field):                                                               1.7%       <3.0%                                         ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Parameter Results and Limits                                                  Comparison for Thick Slide Field Uniformity                                   Parameter Description                                                                              Actual Value                                                                            Limits                                         ______________________________________                                        Coefficient of variation:                                                                          0.6%      <1.0%                                          Maximum % variation (full field):                                                                     2.6%      <6.0%                                       Maximum % Variation (partial field):                                                               1.7%         <3.0%                                       ______________________________________                                    

Strobe Repeatability

Referring now to FIG. 4, FIG. 4 shows an evaluation apparatus used in astrobe repeatability test method of the invention. In one exampleembodiment of the invention a pulsed arc lamp, or strobe 412, forillumination is employed. The strobe transmits light through optics 410.A beam splitter 404 is positioned to receive the light to split thelight into a first beam and a second beam wherein the second beamprovides illumination to condenser lens 402 for a microscopic evaluationof the slide 1. A detector 406 is positioned in an optical path definedby the beam splitter 404 and a second lens 408 to receive the first beamfor providing a detected signal indicative of a first beam intensity.The automated microscope may use the detected signal to adjust forillumination variations. Alternately, a running average of detectedsignals may be obtained and the detected signal may be adjusted for therunning average. Aging of the strobe and drive electronics can cause astrobe to become unstable and vary the energy output from flash toflash. This variation is not unlike variations that may occur in otherillumination sources. The preferred embodiment shown comprises a doublebeam system where detector 406 comprises a PIN diode positioned toreceive light from the strobe as light is split away from the mainoptical path of light directed toward the specimen. As shown, a beamsplitter 404 is placed to split the light before it reaches thespecimen.

Referring now to FIG. 9, a flow diagram of one method of the inventionfor checking strobe repeatability 18 is shown. Data from the strobe maybe collected during the global illumination test at process step 92.From the collected one hundred strobe flashes, the maximum, minimum andcoefficient of variation of strobe output is determined at process step94. In process step 96, the coefficient of variation is compared againsta predetermined coefficient of variation limit. The results areevaluated as follows according to Table 8 at process step 98.

                  TABLE 8                                                         ______________________________________                                        Parameter Results and Limits                                                  Comparison for Strobe Repeatability Test                                      Parameter Description                                                                           Actual Value                                                                            Limits                                            ______________________________________                                        Coefficient of variation:                                                                       0.6%      <1.0%                                             Maximum % variation:                                                                                        <5.0%                                           ______________________________________                                    

Calibration Plate Cleanliness

Referring now to FIG. 10, a flow diagram of one method of the inventionfor checking calibration plate cleanliness 20 is shown. The calibrationand target plate advantageously has a specified clear area forcalibration of pixel gain and offset. At calibration, the calibrationplate is lowered to remove its surfaces from the focal plane of theobjectives. However, because of wide depth of field of somemagnifications, large debris on the calibration plate may be visible.This can cause an erroneous calibration. The calibration plate dirtcheck test is run to check for this condition.

In one method for checking calibration plate cleanliness, a calibrationand test target is introduced into the optical path at process step 102.At process step 104, the calibration and test target is moved about 50microns in both x and y directions from its typical test and calibrationposition. This is done to highlight potentially contaminated areas thatmay be masked by the instrument pixel calibration. An image is collectedand a histogram is generated at process steps 106 and 108 respectively.At step 1002 a coefficient of variation, for both full and partial fieldmaximum variations are computed from the histogram data. At process step1004 a maximum variation and a minimum variation are computed for apredetermined portion of the field-of-view by ignoring outlying pixelsof the histogram so as to provide a measure of the calibration platecleanliness. The results are evaluated by comparing the coefficient ofvariation against a first predetermined limit at step 1006, comparingthe maximum variation against a second predetermined limit at step 1008and comparing the partial field maximum variation against a thirdpredetermined limit in step 1012.

One example of predetermined limits which may be employed are listedbelow.

    ______________________________________                                        Coefficient of variation:  <1.0%                                              Maximum % variation (full field):                                                                               <6.0%                                       Maximums % Variation (partial field):                                                                       <3.0%                                           ______________________________________                                    

Strobe Dropout Monitor

Referring now to FIG. 11, a flow diagram of one method of the inventionfor checking strobe drop out is shown. The method includes the steps ofmaintaining a running average of strobe intensity samples over apredetermined number of samples 1102, comparing each strobe intensitysample against a dropout limit 1104, counting each strobe intensitysample which does not meet the dropout limit as a dropout sample toobtain a plurality of dropout samples 1106, summing the plurality ofdropout samples occurring during a predetermined event to produce adropout sum 1108, and after the predetermined event, comparing thedropout sum to a dropout sum limit 1110.

In operation, occasionally, a strobe lamp will generate arcs that areerratic in position or energy output. This type of behavior can causethe illumination system to produce a field of lower illuminance. In thisembodiment of the invention, the strobe is constantly monitored. Arunning average of the strobe is maintained consisting of 500 samples.All samples taken that exceed 5% of the running average are considereddrop outs. The strobe drop out monitor test sums these dropouts during atray of processing (8 slides), or roughly one hour. After processing atray, the sum is compared to a limit. In this embodiment, the total ofacceptable errant flashes is around 0.03%. The limit is evaluatedagainst a standard as follows: strobe dropouts greater than 5%: <30dropouts per tray.

The invention has been described herein in considerable detail in orderto comply with the Patent Statutes and to provide those skilled in theart with the information needed to apply the novel principles and toconstruct and use such specialized components as are required. However,it is to be understood that the invention can be carried out byspecifically different equipment and devices, and that variousmodifications, both as to the equipment details and operatingprocedures, can be accomplished without departing from the scope of theinvention itself.

For example, this invention comprises of a suite of tests and parametermonitoring scheme to characterize certain facets of illumination. Theabove-described tests refer specifically to a system with a pulsed arclamp and CCD imaging device. However, the concepts may be employed tocontinuous arc lamps, filament lamps, LASER sources, tube cameras, TDIsensors, tube cameras and PIN diodes and photomultiplier tubes.

What is claimed is:
 1. A method of checking illumination uniformity of an optical system illuminated by an illumination system comprising the steps of:(a) acquiring a predetermined number of images of a set of calibration slide areas of varying slide thicknesses with the optical system; (b) determining the illumination uniformity of the optical system from the predetermined number of images; (c) determining a first coefficient of variation and a first coefficient of maximum variation for both a full field and a partial field for a first slide thickness; and (d) determining a second coefficient of variation and a second coefficient of maximum variation, for both a full field and a partial field for a second slide thickness.
 2. The method of claim 1 further comprising the steps of:(a) determining a thin slide coefficient of variation; (b) determining a thin slide maximum percent variation full field; and (c) determining a thin slide maximum percent variation partial field.
 3. The method of claim 1 further comprising the steps of:(a) determining a thick slide coefficient of variation; (b) determining a thick slide maximum percent variation full field; and (c) determining a thick slide maximum percent variation partial field.
 4. A method of checking illumination uniformity of an optical system illuminated by an illumination system comprising the steps of:(a) acquiring a predetermined number of images of a set of calibration slide areas of varying slide thicknesses with the optical system; (b) determining the illumination uniformity of the optical system from the predetermined number of images of the set of calibration slides; (c) determining a thin slide coefficient of variation; (d) determining a thin slide maximum percent variation full field; and (e) determining a thin slide maximum percent variation partial field.
 5. The method of claim 4 further comprising the steps of:(a) introducing a calibration and test target into an optical path of the optical system as part of the step of acquiring a predetermined number of images; (b) determining a first coefficient of variation and a first coefficient of maximum variation for both a full field and a partial field for a first slide thickness; and (c) determining a second coefficient of variation and a second coefficient of maximum variation, for both a full field and a partial field for a second slide thickness.
 6. The method of claim 4 further comprising the steps of:(a) determining a thick slide coefficient of variation; (b) determining a thick slide maximum percent variation full field; and (c) determining a thick slide maximum percent variation partial field.
 7. A method of checking illumination uniformity of an optical system illuminated by an illumination system comprising the steps of:(a) acquiring a predetermined number of images of a set of calibration slide areas of varying slide thicknesses with the optical system; (b) determining the illumination uniformity of the optical system from the predetermined number of images of the set of calibration slides; (c) determining a thick slide coefficient of variation; (d) determining a thick slide maximum percent variation full field; and (e) determining a thick slide maximum percent variation partial field.
 8. The method of claim 7 further comprising the steps of:(a) determining a thin slide coefficient of variation; (b) determining a thin slide maximum percent variation full field; and (c) determining a thin slide maximum percent variation partial field.
 9. An apparatus for checking illumination uniformity of an optical system illuminated by an illumination system comprising:(a) means for acquiring a predetermined number of images of a set of calibration slide areas of varying thicknesses with the optical system, the acquiring means having an image output; (b) means, connected to receive the image output, for determining the illumination uniformity of the optical system from the predetermined number of images; (c) means for determining a first coefficient of variation and a first coefficient of maximum variation for both a full field and a partial field for a first slide thickness; and (d) means for determining a second coefficient of variation and a second coefficient of maximum variation, for both a full field and a partial field for a second slide thickness.
 10. The apparatus of claim 9 further comprising:(a) means for determining a thin slide coefficient of variation; (b) means for determining a thin slide maximum percent variation full field; and (c) means for determining a thin slide maximum percent variation partial field.
 11. The apparatus of claim 9 further comprising:(a) means for determining a thick slide coefficient of variation; (b) means for determining a thick slide maximum percent variation full field; and (c) means for determining a thick slide maximum percent variation partial field.
 12. The apparatus of claim 9 comprising a means for introducing a calibration and test target into an optical path of the optical system connected to cooperate with the means for acquiring a predetermined number of images.
 13. The apparatus of claim 9 wherein the means for acquiring a predetermined number of images further comprises an automated microscope.
 14. The apparatus of claim 13 wherein the means for acquiring a predetermined number of images further comprises a CCD camera within the optical path.
 15. The apparatus of claim 13 wherein the illumination system comprises a strobe light.
 16. The apparatus of claim 9 wherein the illumination system comprises a strobe light. 